Xiayuan Liang

Inhibition of Mer and Axl Receptor Tyrosine Kinases in Astrocytoma Cells Leads to Increased Apoptosis and Improved Chemosensitivity

Astrocytomas account for the majority of malignant brain tumors diagnosed in both adult and pediatric patients. The therapies available to treat these neoplasms are limited, and the prognosis associated with high-grade lesions is extremely poor. Mer (MerTK) and Axl receptor tyrosine kinases (RTK) are expressed at abnormally high levels in a variety of malignancies, and these receptors are known to activate strong antiapoptotic signaling pathways that promote oncogenesis. In this study, we found that Mer and Axl mRNA transcript and protein expression were elevated in astrocytic patient samples and cell lines. shRNA-mediated knockdown of Mer and Axl RTK expression led to an increase in apoptosis in astrocytoma cells. Apoptotic signaling pathways including Akt and extracellular signal–regulated kinase 1/2, which have been shown to be activated in resistant astrocytomas, were downregulated with Mer and Axl inhibition whereas poly(ADP-ribose) polymerase cleavage was increased. Furthermore, Mer and Axl shRNA knockdown led to a profound decrease of astrocytoma cell proliferation in soft agar and a significant increase in chemosensitivity in response to temozolomide, carboplatin, and vincristine treatment. Our results suggest Mer and Axl RTK inhibition as a novel method to improve apoptotic response and chemosensitivity in astrocytoma and provide support for these oncogenes as attractive biological targets for astrocytoma drug development. Mol Cancer Ther; 9(5); 1298–307. ©2010 AACR.

Natural killer cell neoplasms.

Natural killer (NK) cell tumors are an uncommon and heterogeneous group of disorders. The World Health Organization (WHO) classified mature NK cell neoplasms into 2 types: 1) extranodal NK cell lymphoma, nasal type and 2) aggressive NK cell leukemia. The mature NK cell tumors are prevalent in Asia and Central and South America. These tumors show polymorphic neoplastic infiltrate with angioinvasion and/or angiodestruction, cytoplasmic azurophilic granules, CD2‐positive (CD2+)/CD3‐negative (CD3−)/cCD3ε+/CD56+ phenotype, and strong association with Epstein‐Barr virus (EBV). Loss of chromosomes 6q, 11q, 13q, and 17p are recurrent aberrations. Although blastic NK cell lymphoma, currently referred to as CD4+/CD56+ hematodermic neoplasm, also was included in the NK cell lymphoma category in the WHO classification scheme, existing evidence indicates a plasmacytoid dendritic cell derivation as opposed to an NK cell origin. Recently, rare cases of CD56+ immature lymphoid tumors have been reported in the literature. These tumors are characterized by blastic appearance, CD3−/CD4−/CD56+/CD13−/CD33− phenotype, T‐cell receptor and immunoglobulin genes in germline configuration, and no evidence of EBV, suggesting a true immature NK cell derivation. For this article, the authors reviewed the recent concepts and progress in clinicopathologic features, pathogenesis, genetic characteristics, diagnosis, differential diagnosis, treatment approaches, and outcomes of all subtypes of NK cell neoplasms. Cancer 2008.

Ectopic Expression of the Proto-oncogene Mer in Pediatric T-Cell Acute Lymphoblastic Leukemia

Purpose: The Mer receptor tyrosine kinase, cloned from a B-lymphoblastoid library, is the mammalian orthologue of the chicken retroviral oncogene v-eyk and sends antiapoptotic and transforming signals when activated. To determine if Mer expression is ectopic in T-cell acute lymphoblastic leukemia (ALL) and potentially important in leukemogenesis, we analyzed Mer expression in normal human thymocytes and lymphocytes and in pediatric ALL patient samples. Experimental Design: Reverse transcription-PCR, flow cytometry, and immunohistochemistry were used to determine expression of Mer in sorted human thymocyte populations, lymphocytes, and lymphocytes activated by phytohemagglutinin or phorbol 12-myristate 13-acetate/ionophore. Mer expression in 34 T-cell ALL (T-ALL) patient samples was evaluated by reverse transcription-PCR, and Mer protein expression in a separate cohort of 16 patient samples was assayed by flow cytometry and Western blot. Results: Mer expression was absent in normal thymocytes or lymphocytes, and in T cells activated with phytohemagglutinin or phorbol 12-myristate 13-acetate/ionophore. In contrast, Jurkat cells and T-ALL patient samples expressed unique 180 to 185 kDa Mer protein glycoforms. Substantial Mer RNA levels were principally observed in a subset of T-ALL patient samples that expressed B220 (P = 0.004) but lacked surface expression of CD3 (P = 0.02) and CD4 (P = 0.006), a phenotypic profile consistent with immature lymphoblasts. In addition, 8 of 16 T-ALL patient samples had Mer protein detected by flow cytometry and Western blot. Conclusions: Transforming Mer signals may contribute to T-cell leukemogenesis, and abnormal Mer expression may be a novel therapeutic target in pediatric ALL therapy.

Lymphoblastic leukemia/lymphoma in mice overexpressing the Mer (MerTK) receptor tyrosine kinase

Mer (MerTK) is a receptor tyrosine kinase important in platelet aggregation, as well as macrophage cytokine secretion and clearance of apoptotic cells. Mer is not normally expressed in thymocytes or lymphocytes; however, ectopic Mer RNA transcript and protein expression is found in a subset of acute lymphoblastic leukemia cell lines and patient samples, suggesting a role in leukemogenesis. To investigate the oncogenic potential of Mer in vivo, we created a transgenic mouse line (MerTg) that expresses Mer in the hematopoietic lineage under control of the Vav promoter. Ectopic expression and activation of the transgenic Mer protein was demonstrated in lymphocytes and thymocytes of the MerTg mice. At 12–24 months of age, greater than 55% of the MerTg mice, compared to 12% of the wild type, developed adenopathy, hepatosplenomegaly, and circulating lymphoblasts. Histopathological analysis and flow cytometry were consistent with T-cell lymphoblastic leukemia/lymphoma. Mer may contribute to leukemogenesis by activation of Akt and ERK1/2 anti-apoptotic signals, which were upregulated in MerTg mice. Additionally, a significant survival advantage was noted in MerTg lymphocytes compared to wild-type lymphocytes after dexamethasone treatment. These data suggest that Mer plays a cooperative role in leukemogenesis and may be an effective target for biologically based leukemia/lymphoma therapy.

Aberrant Mer receptor tyrosine kinase expression contributes to leukemogenesis in acute myeloid leukemia.

Acute myeloid leukemia (AML) continues to be extremely difficult to treat successfully, and the unacceptably low overall survival rates mandate that we assess new potential therapies to ameliorate poor clinical response to conventional therapy. Abnormal tyrosine kinase activation in AML has been associated with poor prognosis and provides strategic targets for novel therapy development. We found that Mer receptor tyrosine kinase was over-expressed in a majority of pediatric (29/36, 80%) and adult (10/10, 100%) primary AML patient blasts at the time of diagnosis, and 100% of patient samples at the time of relapse. Mer was also found to be expressed in 12 of 14 AML cell lines (86%). In contrast, normal bone marrow myeloid precursors expressed little to no Mer. Following AML cell line stimulation with Gas6, a Mer ligand, we observed activation of prosurvival and proliferative signaling pathways, including phosphorylation of ERK1/2, p38, MSK1, CREB, ATF1, AKT and STAT6. To assess the phenotypic role of Mer in AML, two independent short-hairpin RNA (shRNA) constructs were used to decrease Mer expression in the AML cell lines Nomo-1 and Kasumi-1. Reduction of Mer protein levels significantly increased rates of myeloblast apoptosis two to threefold in response to serum starvation. Furthermore, myeloblasts with knocked-down Mer demonstrated decreased colony formation by 67–87%, relative to control cell lines (P<0.01). NOD-SCID-gamma mice transplanted with Nomo-1 myeloblasts with reduced levels of Mer had a significant prolongation in survival compared with mice transplanted with the parental or control cell lines (median survival 17 days in parental and control cell lines, versus 32–36 days in Mer knockdown cell lines, P<0.0001). These data suggest a role for Mer in acute myeloid leukemogenesis and indicate that targeted inhibition of Mer may be an effective therapeutic strategy in pediatric and adult AML.

Proteomic Analyses of Human Plasma: Venus versus Mars

BACKGROUND: Plasma is vital for the resuscitation of injured patients and to restore necessary procoagulants, especially Factors (F)II, FV, FVII, FX, and FXIII; however, female plasma has been implicated in the majority of transfusion‐related acute lung injury (TRALI) cases and male‐only plasma transfusion regimens have significantly decreased the incidence of TRALI. Little is known about the human plasma proteome, and no comparisons have been made between male and female plasma; therefore, we hypothesize that there are significant differences between plasma from male and female donors.

Mer receptor tyrosine kinase is a novel therapeutic target in pediatric B-cell acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is currently treated with an intense regimen of chemotherapy yielding cure rates near 80%. However, additional changes using available drugs are unlikely to provide significant improvement in survival. New therapies are warranted given the risk of severe therapy-associated toxicities including infertility, organ damage, and secondary malignancy. Here, we report ectopic expression of the receptor tyrosine kinase Mer in pediatric B-cell ALL. Inhibition of Mer prevented Erk 1/2 activation, increased the sensitivity of B-ALL cells to cytotoxic agents in vitro by promoting apoptosis, and delayed disease onset in a mouse model of leukemia. In addition, we discovered cross-talk between the Mer and mammalian target of rapamycin (mTOR) signaling pathways. Our results identify Mer as a novel therapeutic target in ALL and suggest that inhibitors of Mer will interact synergistically with currently used therapies. This strategy may allow for dose reduction resulting in decreased toxicity and increased survival rates. Mer is aberrantly expressed in numerous other malignancies suggesting that this approach may have broad applications.

E2A - ZNF384 and NOL1 - E2A fusion created by a cryptic t(12;19)(p13.3; p13.3) in acute leukemia

A 5-year-old boy who initially presented with ALL and relapsed 4 months later with AML was found to have an add(19) in the leukemia cells. FISH revealed that the add(19) was really a cryptic t(l2;l9)(p13.3;p13.3) interrupting E2A (TCF3). Nucleotide sequences of cloned genomic fragments with the E2A rearrangements revealed that the der(12) contained E2A joined to an intron of the NOLI (p120) gene. Reverse transcriptase (RT)–PCR of patient lymphoblast RNA showed expression of in-frame fusion cDNAs consisting of most of NOL1 fused to the 3′ portion of E2A that encoded part of the second transcriptional activation domain and the DNA binding and protein dimerization motifs. The reciprocal der(19) E2A genomic rearrangements included 5′ regions of E2A joined to an intron of the ZNF384 (NMP4, CIZ) gene, located approximately 450 kb centromeric to NOL1 on chromosome 12. RT–PCR showed expression of in-frame E2A-ZNF384 fusion cDNAs. To our knowledge, this is the second report of a chromosome translocation in leukemia resulting in two different gene fusions. This is the first report of expression of E2A fusion protein that includes the DNA binding and protein dimerization domains due to a more proximal break in E2A compared to those described previously.

Thymoma in Children: Report of 2 Cases and Review of the Literature

Thymoma is an uncommon and slow-growing neoplasm. It is derived from thymic epithelial cells and comprises about 20% to 30% of mediastinal masses in adults, but only about 1% in pediatric patients. Patients usually present with mass-associated respiratory symptoms, superior vena cava syndrome, or paraneoplastic syndrome including myasthenia gravis, pure red cell aplasia, or acquired hypogammaglobulinemia, and connective tissue disorders. Due to the limited number of cases, knowledge, and experience with thymoma in pediatric patients, the diagnosis and treatment are very challenging for this age group. In this article, we report 2 cases of thymoma in childhood and provide a comprehensive review and analysis of the reported pediatric cases in the past 30 years (total of 32 cases). We found that patients younger than age 10 years were predominantly male (M:F = 6:1) and had advanced tumor stage more frequent than patients older than age 10 (P = .03). There were also significant associations of male sex with more advanced tumor stage and less favorable outcome (P = .03). These findings suggest that age and sex may be additional potential prognostic contributors in pediatric patients with thymoma. The clinicopathologic features, differential diagnosis, and current therapeutic recommendations of this uncommon tumor in pediatric patients are also addressed.

Activated Platelets in Heparinized Shed Blood: The “Second-Hit” of Acute Lung Injury in Trauma/Hemorrhagic Shock Models

ABSTRACT The return of heparinized shed blood (SB) in trauma/hemorrhagic shock (T/HS) models remains controversial because of potential anti-inflammatory properties. Although ubiquitous as an anticoagulant, heparin is ineffective on cellular coagulation as an antithrombotic agent. Therefore, we hypothesized that returning heparinized SB would paradoxically enhance acute lung injury (ALI) after T/HS because of the infusion of activated platelets. Sprague-Dawley rats, anesthetized with pentobarbital, underwent laparotomy and hemorrhage-induced shock (MAP of 30 mmHg × 45 min). Animals were resuscitated with a combination of normal saline and returned SB. Shed blood was collected in either 80 U/kg of heparin, 800 U/kg of heparin, or citrate or diluted 1:8 with normal saline. An additional group of animals were pretreated with a platelet P2Y12 receptor antagonist (clopidogrel) before T/HS. Bronchoalveolar lavage, lung myeloperoxidase assays, pulmonary immunofluorescence, and blood smears were conducted. Bronchoalveolar lavage protein increased in animals resuscitated with heparinized SB (T/HS + 80 U/kg Hep 1.62 ± 0.29, T/HS + 800 U/kg Hep 1.30 ± 0.15 vs. T/SS 0.51 ± 0.16 and T/HS Citrate 0.7 ± 0.09) (P < 0.0001). Blood smears and platelet function assays revealed platelet aggregates and increased platelet activation. Animals pretreated with a platelet P2Y12 receptor antagonist were protected from postinjury ALI (P < 0.0001). Animals with return of SB had increased pulmonary polymorphonuclear leukocyte sequestration (P < 0.0001). Pulmonary immunofluorescence demonstrated microthrombi only in the T/HS group receiving heparinized SB (P < 0.0001). The return of heparinized SB functions as a “second hit” to enhance ALI, with activated platelets propagating microthrombi and pulmonary polymorphonuclear leukocyte recruitment.